Peptides acting as both GLP-1 receptor agonists and glucagon receptor antagonists and their pharmacological methods of use

ABSTRACT

The invention provides polypeptides that act both as an agonist of the GLP-1 receptor and an antagonist of the glucagon receptor. Such polypeptides are useful for treating individuals with type 2 diabetes or other metabolic disorders.

This application is a divisional application of U.S.S.N. 10/265,345,filed Oct. 3, 2002, now U.S. Pat. No. 6,864,069.

FIELD OF THE INVENTION

This invention relates to newly identified polypeptides that act both asan agonist of the GLP-1 receptor and an antagonist of the glucagonreceptor and the use of such polypeptides for therapeutic purposes. Moreparticularly, polypeptides of the present invention are useful instimulating the release of insulin from pancreatic beta cells in aglucose-dependent manner and reducing glucagon-mediated secretion ofglucose from the liver, thereby providing a treatment option for thoseindividuals afflicted with a metabolic disorder such as diabetes,hyperglycemia, impaired fasting glucose, impaired glucose tolerance,prediabetic states, and obesity.

BACKGROUND OF THE RELATED ART

Diabetes is characterized by impaired insulin secretion manifestingitself among other things by an elevated blood glucose level in thediabetic patient. Underlying defects lead to a classification ofdiabetes into two major groups: type I diabetes (or insulin dependentdiabetes mellitus, IDDM), which arises when patients lackinsulin-producing beta-cells in their pancreatic glands, and type 2diabetes (or non-insulin dependent diabetes mellitus, NIDDM), whichoccurs in patients with an impaired beta-cell insulin secretion and/oralterations in insulin action.

Type 1 diabetic patients are currently treated with insulin, while themajority of type 2 diabetic patients can be treated with agents thatstimulate beta-cell function or with agents that enhance the tissuesensitivity of the patients towards insulin. Over time, almost one-halfof type 2 diabetic subjects lose their response to these agents and thenmust be placed on insulin therapy. The drugs presently used to treattype 2 diabetes include alpha-glucosidase inhibitors (PRECOSE®,VOGLIBOSE™, and MIGLITOL®), insulin sensitizers (e.g., Avandia™, Actos™and Rezulin™), insulin secretagogues (sulfonylureas (“SFUs”) and otheragents that act by the ATP-dependent K⁺ channel), and GLUCOPHAGE™(metformin HCl).

Alpha-glucosidase inhibitors. Alpha-glucosidase inhibitors reduce theexcursion of postprandial glucose by delaying the absorption of glucosefrom the gut. These drugs are safe and provide treatment for mild tomoderately affected diabetic subjects. However, gastrointestinal sideeffects have been reported in the literature and limit theireffectiveness.

Insulin sensitizers. Insulin sensitizers are drugs that enhance thebody's response to insulin. Thiozolidinediones such as Avandia™(rosiglitazone) and Actos™ activate peroxisome proliferator-activatedreceptor (PPAR) gamma and modulate the activity of a set of genes thathave not been well described. Hepatic effects (e.g., drug inducedhepatotoxicity and elevated liver enzyme levels) do not appear to be asignificant problem in patients using Avandia™ and Actos™. Even so,liver enzyme testing is recommended every two months in the first yearof therapy and periodically thereafter. Avandia™ and Actos™ treatmentsare associated with fluid retention, edema, and weight gain. Avandia™ isnot indicated for use with insulin because of concern about congestiveheart failure. Rezulin™ (troglitazone), the first drug in this class,was withdrawn because of elevated liver enzyme levels and drug-inducedhepatotoxicity.

Insulin secretagogues. Sulfonylureas (SFUs) and the non-sulfonylureas,Nateglinide and Pepaglinide act through the ATP-dependent potassiumchannel to cause glucose-independent insulin secretion. These drugs arestandard therapy for type 2 diabetics that have mild to moderate fastinghyperglycemia. The insulin secretagogues have limitations that include apotential for inducing hypoglycemia, weight gain, and high primary andsecondary failure rates. Ten to 20% of initially treated patients failto show a significant treatment effect (primary failure). Secondaryfailure is demonstrated by an additional 20-30% loss of treatment effectafter six months of treatment with insulin secretagogues. Insulintreatment is required in 50% of the insulin secretagogues respondersafter 5-7 years of therapy (Scheen et al, Diabetes Res. Clin. Pract.6:533-543, 1989). Nateglinide and Pepaglinide are short-acting drugsthat need to be taken three times a day. They are used only for thecontrol of post-prandial glucose and not for control of fasting glucose.

GLUCOPHAGE™ is a biguanide that lowers blood glucose by decreasinghepatic glucose output and increasing peripheral glucose uptake andutilization. The drug is effective at lowering blood glucose in mildlyand moderately affected subjects and does not have a side effect ofweight gain or a potential to induce hypoglycemia. However, GLUCOPHAGE™has a number of side effects, including gastrointestinal disturbancesand lactic acidosis. GLUCOPHAGE™ is contraindicated in diabetics overthe age of 70 and in subjects with impaired renal or liver function.Finally, GLUCOPHAGE™ has the same primary and secondary failure rates asthe insulin secretagogues.

Insulin treatment is instituted after diet, exercise, and oralmedications have failed to control blood glucose adequately. Thistreatment has several drawbacks: it is an injectable, it can producehypoglycemia, and it can cause weight gain. The possibility of inducinghypoglycemia with insulin limits the extent that hypoglycemia can becontrolled.

Problems with current treatments necessitate new therapies to treat type2 diabetes. In particular, new treatments to retain normal (i.e.,glucose-dependent) insulin secretion are needed. Given glucagon-likepeptide-1's (“GLP-1”) role in promoting glucose-regulated insulinsecretion in the pancreas, GLP-1 receptor agonists are potentiallyvaluable in the treatment of such diseases. Moreover, glucagon receptorantagonists should prove valuable in treating type 2 diabetes givenglucagon's role in elevating plasma glucose by stimulating hepaticglycogenolysis and gluconeogenesis.

GLP-1 and glucagon are members of a family of structurally relatedpeptide hormones, the glucagon/secretin family. Within this family,GLP-1 (7-36) and GLP-1 (7-37) (30 amino acids and 31 amino acids,respectively) and glucagon (30 amino acids) constitute a highlyhomologous set of peptides. In addition, these two hormones originatefrom a common precursor, preproglucagon which, upon tissue-specificprocessing, leads to production of GLP-1 predominantly in the intestineand glucagon in the pancreas. The receptors for these two peptides arehomologous (58% identity) and belong to the family of G-protein coupledreceptors.

GLP-1 and glucagon both play major roles in overall glucose homeostasis.GLP-1 lowers plasma glucose concentrations mediated by glucose dependentinsulin secretion, whereas glucagon increases plasma glucoseconcentrations. Given the important roles of both GLP-1 and glucagon inmaintaining normal blood glucose concentrations, there has beenconsiderable interest in the identification of GLP-1 receptor agonistsand glucagon receptor antagonists. Clinical studies have demonstratedthe ability of GLP-1 infusion to promote insulin secretion and tonormalize plasma glucose in diabetic subjects. However, GLP-1 is rapidlydegraded and has a very short half-life in the body. Furthermore, GLP-1causes gut motility side effects at or near its therapeutic doses.Therefore, GLP-1 itself has significant limitations as a therapeuticagent, and modified versions of the peptide with enhanced stability arebeing pursued. Non-peptide agonists of the GLP-1 receptor have not beendescribed to date.

Peptide analogs of glucagon have been identified which act as glucagonantagonists and reduce hyperglycemia in diabetic rats. However, nopeptide glucagon antagonist has moved beyond preclincal development. Anumber of structurally diverse non-peptide glucagon receptor antagonistshave been reported in the scientific and patent literature. However,attempts to identify small molecule inhibitors of the glucagon receptorhave met with limited success in vivo. The only antagonist of glucagonaction known to be active in a clinical study is a compound identifiedas BAY 27-9955. A potential side effect of glucagon antagonism ishypoglycemia.

Because of the potential side effects associated with administeringeither a GLP-1 receptor agonist or a glucagon receptor antagonist alone,a combination therapy would have an advantage of maintaining the desiredlowering of blood glucose while reducing the side effects.Co-administration, however, requires a single formulation and deliveryapproach that yields appropriate pharmokinetic profiles for bothpeptides. This could be a major obstacle to the development of such atherapeutic.

Based on the foregoing, considerable potential exists for a singletherapeutic peptide functioning as both a GLP-1 agonist and a glucagonantagonist in vivo.

SUMMARY OF THE INVENTION

This invention provides novel polypeptides that function both as anagonist of the GLP-1 receptor and an antagonist of the glucagon receptorand which are effective in the treatment of diseases and conditions thatcan be ameliorated by agents having both GLP-1 receptor agonist andglucagon receptor antagonist activity. Polypeptides of the presentinvention provide a new therapy for patients with, for example,metabolic disorders, such as those resulting from decreased endogenousinsulin secretion, in particular type 2 diabetics, or for patients withimpaired glucose tolerance, a prediabetic state that has a mildalteration in insulin secretion or impaired fasting glucose, or obesity.

One aspect of the invention is a polypeptide selected from the groupconsisting of SEQ ID NOS:6-32, as well as fragments, derivatives andvariants of polypeptides that function as both an agonist of the GLP-1receptor and an antagonist of the glucagon receptor at substantially thesame level as the polypeptides shown in SEQ ID NOS:6-32 (collectively,“polypeptides of the invention”).

Other embodiments of the invention include polynucleotides that encodepolypeptides of the invention and the attendant vectors and host cellsnecessary to recombinantly express the polypeptides.

Still other embodiments of the invention provide methods of treatingdiabetes and/or other diseases or conditions affected by polypeptides ofthe invention in mammals, including humans. The methods involveadministering a therapeutically effective amount of any of thepolypeptides of the present invention to a mammal.

The invention also provides recombinant and synthetic methods of makingpolypeptides of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a restriction map of a typical plasmid containing aGST-peptide fusion polynucleotide coding sequence.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides novel polypeptides, as well as fragments,derivatives variants, and analogs thereof that function as both anagonist of the GLP-1 receptor and an antagonist of the glucagonreceptor. Polypeptides of the invention function in vivo as both GLP-1receptor agonists and glucagon receptor antagonists in the preventionand/or treatment of such diseases or conditions as diabetes,hyperglycemia, impaired glucose intolerance, impaired fasting glucose,and obesity.

GLP-1 and glucagon are members of a family of structurally relatedpeptide hormones, the glucagon/secretin family. The stacking alignmentbelow shows the primary structural relationships:

(SEQ ID NO: 1) Glucagon HSQGTFTSDYSKYLEGQAAKEFIAWLVKGR (SEQ ID NO: 2)GLP-1 (7–36) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂ (SEQ ID NO: 3) GLP-1(7–37) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSingle-letter abbreviations for amino acids can be found in Zubay,Biochemistry 2d ed., 1988, MacMillan Publishing, New York, p. 33. Thesepolypeptides play a role in overall glucose homeostasis: GLP-1 lowersplasma glucose concentrations, whereas glucagon increases plasma glucoseconcentrations.

Given GLP-1 's role in promoting glucose-regulated insulin secretion inthe pancreas, GLP-1 receptor agonists are potentially valuable in thetreatment of metabolic disorders and other diseases. Moreover, glucagonreceptor antagonists should also prove valuable in treating diseasegiven glucagon's role in elevating plasma glucose by stimulating hepaticglycogenolysis and gluconeogenesis. However, these facts alone do notguarantee glucose reduction in vivo without inducing significant sideeffects.

The invention provides new polypeptides that are both GLP-1 receptoragonists and glucagon receptor antagonists. Without being bound totheory, we believe that polypeptides of the invention are capable ofreducing glucose levels in vivo by stimulating insulin release frompancreatic beta cells in a glucose-dependent manner, while at the sametime reducing glucagon-mediated secretion of glucose from the liver.

GLP-1 Receptor Agonist and Glucagon Receptor Antagonist Polypeptides

Polypeptides of the invention function both as a GLP-1 receptor agonistand a glucagon receptor antagonist. The GLP-1 receptor agonist componentof such polypeptides activates the GLP-1 receptor in one or more invitro or in vivo assays for GLP-1 receptor activation. Examples of suchassays include, but are not limited to, in vitro assays for induction ofcAMP in RINm5F cells, in vitro assays for induction of insulin secretionfrom pancreatic β-cells, in vivo assays for reduction in plasma glucoselevels, and in vivo assays for elevation in plasma insulin levels asdescribed in the specific examples below.

Polypeptides of the invention also demonstrate glucagon receptorantagonist activity in one or more in vitro or in vivo assays forinhibition of glucagon receptor activation. Examples of such assaysinclude, but are not limited to, in vitro assays to measure inhibitionof glucagon-mediated increase in cellular cAMP, in vitro assays tomeasure inhibition of glucagon-mediated glucose release from, forexample, cultured hepatocytes, or in vivo assays for glucagon stimulatedglucose production as described in the specific examples below.

Preferred polypeptides of the invention are selected from the groupconsisting of (1) SEQ ID NOS:6-32 and (2) fragments, derivatives,variants, and analogs thereof that function as both an agonist of theGLP-1 receptor and an antagonist of the glucagon receptor atsubstantially the same level as any of the polypeptides shown in SEQ IDNOS:6-32.

Polypeptides of the present invention may be naturally-occurringpolypeptides, recombinant polypeptides, or synthetic polypeptides.

Fragments, Derivatives, Variants, and Analogs

Fragment, derivative, variant, and analog polypeptides retainsubstantially the same biological function or activity as, for example,polypeptides shown in SEQ ID NOS:6-32. “Substantially the samebiological function or activity” is about 30% to 100% (i.e., 30, 40, 50,60, 70, 80, 90, or 100%) or more of the same biological activity of thefull-length polypeptide to which it is compared.

Derivatives

Derivatives include polypeptides of the invention that have beenchemically modified to provide an additional structure and/or function.For example, polyethylene glycol (PEG) or a fatty acid can be added to apolypeptide to improve its half-life. Fusion polypeptides which confertargeting specificity or an additional activity also can be constructed,as described in more detail below.

Derivatives can be modified by either a natural process, such asposttranslational processing, or by chemical modification techniques,both of which are well known in the art. Modifications can occuranywhere in a polypeptide, including the peptide backbone, the aminoacid side-chains, and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present to thesame or varying degrees at several sites in a given polypeptide. Also, avariant may contain one or more different types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.

Other chemical modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formulation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, T. E. Creighton, PROTEINS, STRUCTURE AND MOLECULARPROPERTIES, 2nd ed., W.H. Freeman and Company, New York (1993);POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol 182:626-46 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62, 1992).

Derivatives also include mature polypeptides that have been fused withanother polypeptide, such as for example human serum albumin, to improvetheir pharmacokinetic profile. Fusion of two polypeptides can beaccomplished by any means known to one skilled in the art. For example,a DNA encoding human serum albumin and a DNA sequence encoding apolypeptide of the invention can be cloned into any mammalian expressionvector known to one skilled in the art. Location of a polypeptide of theinvention N-terminal to the other polypeptide is preferred, because itappears that a free N-terminal histidine is required for GLP-1 receptoractivity (Kawa, Endocrinology Apr;124(49): 1768-73, 1989). The resultingrecombinant fusion protein can then be expressed by transforming asuitable cell line, such as HKB or CHO, with the vector and expressingthe fusion protein.

Preferred derivatives include polypeptides of the invention (SEQ IDNOS:6-32) to which a PEG moiety or a fatty acid moiety has beenattached. The PEG moiety can be, for example, a PEG with a molecularweight greater than 22 kDa, preferably a molecular weight of between 25kDa and 100 kDa (e.g., 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 kDa), and more preferably a molecular weight of between 35kDa and 45 kDa (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45kDa). Examples of such PEGylated polypeptides are those that contain a22 kDa PEG moiety attached to the cysteine residue at position 31 of SEQID NOS:19 or 25. See SEQ ID NOS:20 and 26 and Table 2. Alternatively, a43 kDa PEG moiety can be attached to the cysteine residue at position 31of SEQ ID NO:25. See SEQ ID NO:27 and Table 2. A PEG moiety can be addedto a cysteine residue of polypeptides of the invention by methods wellknown in the art, for example, see Example 19.

Other preferred derivatives have a fatty acid moiety attached to thepolypeptide. The fatty acid moiety can be, for example, a fatty acidbetween C₁₂ and C₂₀, preferably C₁₄ and C₁₈, and most preferably a C₁₆fatty acid. Examples of such polypeptides are SEQ ID NOS:28-32, whichcontain a C₁₆ (palmitate) fatty acid moiety attached to a lysine residuewithin the peptide. See Table 2. A fatty acid moiety can be added to alysine residue of polypeptides of the invention by methods well known inthe art, for example, fatty acid acylation (Knudsen et al., J. Med.Chem. 43:1664-1669, 2000).

Variants

Variants are polypeptides on the invention that have or more amino acidsequence changes with respect to the amino acid sequences shown in SEQID NOS:6-32. Variants also can have amino acids joined to each other bymodified peptide bonds, i.e., peptide isosteres, and may contain aminoacids other than the 20 naturally occurring amino acids.

Preferably, variants contain one or more conservative amino acidsubstitutions (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions),preferably at nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from a wild-type sequenceof a protein without altering its biological activity, whereas an“essential” amino acid residue is required for biological activity. Aconservative amino acid substitution is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Non-conservativesubstitutions would not be made for conserved amino acid residues or foramino acid residues residing within a conserved protein domain.

Conservative amino acid substitutions are preferably at positions 11,12, 16, 17, or 18 of the consensus polypeptide shown SEQ ID NO:34.Position 11 preferably is R, S, A, K, G, or T and more preferably R, A,G, or S. Position 12 preferably is K, N, R, H, A, S or Q and morepreferably K, A, S, or N. Position 16 preferably is K, R, V, I, L, M, F,W, Y, A, S, T, N, Q, G, or H, and more preferably K, V, I, F, A, S, orN. Position 17 preferably is D, E, H, K, R, F, I, L, M, Y, V, W, A, S,T, N, Q, or G, and more preferably R, A, L, M, V, S, H, E, or Q.Position 18 preferably is K, R, F, I, L, Y, V, M, A, G, or H and morepreferably K, R, F, I, L, Y or A. All possible combinations ofsubstitutions at positions 11, 12, 16, 17, and 18, including nosubstitution at any one, two, three, or four of these positions, arespecifically envisioned.

Variants also include polypeptides that differ in amino acid sequencedue to mutagenesis. Variants that function as both GLP-1 receptoragonists and glucagon receptor antagonists can be identified byscreening combinatorial libraries of mutants, for example, mutants ofpolypeptides with conservative substitutions at 1 or more positions(i.e., at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions) can be screenedfor GLP-1 receptor agonist activity and glucagon receptor antagonistactivity using methods well known in the art and described in thespecific examples below.

Analogs

An analog includes a propolypeptide, wherein the propolypeptide includesan amino acid sequence of a polypeptide of the invention. Activepolypeptides of the invention can be cleaved from the additional aminoacids in the propolypeptide molecule by natural, in vivo processes or byprocedures well known in the art, such as by enzymatic or chemicalcleavage.

Fragments

A fragment is less than a full-length polypeptide of the invention,including a full-length derivative, variant, or analog, which has GLP-1receptor agonist and glucagon receptor antagonist activity.

Polynucleotides

Any polynucleotide sequence that encodes a polypeptide of the inventioncan be used to express the polypeptide. Polynucleotides can consist onlyof a coding sequence for a polypeptide or can include additional codingand/or non-coding sequences.

Polynucleotide sequences encoding a polypeptide of the invention can besynthesized in whole or in part using chemical methods well known in theart (see, for example, Caruthers et al., Nucl. Acids Res. Symp. Ser.215-23, 1980; Horn et al., Nucl. Acids Res. Symp. Ser. 225-32, 1980).The polynucleotide that encodes the polypeptide can then be cloned intoan expression vector to express the polypeptide or into a cloningvector, to propagate the polynucleotide.

As will be understood by those of skill in the art, it may beadvantageous to produce the polypeptide-encoding nucleotide sequencespossessing non-naturally occurring codons. For example, codons preferredby a particular prokaryotic or eukaryotic host can be selected toincrease the rate of polypeptide expression or to produce an RNAtranscript having desirable properties, such as a half-life, which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter the polypeptide-encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe polypeptide or mRNA product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides canbe used to engineer the nucleotide sequences. For example, site-directedmutagenesis can be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

Vectors

The present invention also includes cloning and expression vectorscomprising one or more nucleotide sequences encoding a polypeptide ofthe invention. The nucleotide sequence can be inserted in a forward orreverse orientation. A DNA sequence may be inserted into a vector by avariety of procedures. In general, a DNA sequence is inserted into anappropriate restriction endonuclease site by procedures known in the artand described in Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2d ed., (Cold Spring Harbor, N.Y., 1989). Such procedures andothers are deemed to be within the scope of those skilled in the art.

Examples of cloning vectors include, but are not limited to pBR322,pUC18, pUC19, pSport, and pCRII.

In a preferred aspect of this embodiment, an expression vector furthercomprises regulatory sequences, including, for example, a promoter,operably linked to the coding sequence. Large numbers of suitableexpression vectors and promoters are known to those of skill in the artand are commercially available. The following expression vectors areprovided by way of example. Bacterial expression vectors include, butare not limited to, pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript,psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a(Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia).Eukaryotic expression vectors include, but are not limited to, pWLneo,pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, PSVL(Pharmacia). However, any other cloning or expression vector may be usedas long as it is replicable and viable in the desired host. Promoterregions can be selected from any desired gene using CAT (chloramphenicoltransferase) expression vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include laci, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of an appropriate vector and promoter iswell within the level of ordinary skill in the art.

An expression vector also can contain a ribosome binding site fortranslation initiation, a transcription terminator, and appropriatesequences for amplifying expression. Expression vectors can contain agene to provide a phenotypic trait for selection of transformed hostcells, such as dihydrofolate reductase or neomycin resistance for aeukaryotic cell culture, or such as tetracycline or ampicillinresistance for culture in E. coli.

Libraries

In one embodiment, a library of variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A library of variants can be produced, for example, byenzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential variant amino acidsequences is expressible as individual polypeptides or, alternatively,as a set of larger fusion proteins (for example, for phage display)containing the set of sequences therein.

There are a variety of methods that can be used to produce libraries ofpotential variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential analog sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, Tetrahedron39:3 (1983); Itakura et al., Annu. Rev. Biochem. 53:323 (1984); Itakuraet al., Science 198:1056 (1984); Ike et al., Nucleic Acid Res. 11:477,1983).

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of polypeptides of theinvention. The most widely used techniques, which are amenable to highthrough-put analysis for screening large gene libraries, typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique that enhances the frequency of functionalmutants in the libraries, can be used in combination with the screeningassays to identify the desired variants.

Host Cells

The present invention also provides host cells containing theabove-described vectors. The host cell can be a higher eukaryotic cell,such as a mammalian cell, or a lower eukaryotic cell, such as a yeastcell. Alternatively, the host cell can be a prokaryotic cell, such as abacterial cell.

Host cells can be genetically engineered (transduced, transformed ortransfected) with cloning or expression vectors of the invention. Thevector may be, for example, in the form of a plasmid, a viral particle,or a phage. Engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters orselecting transformants. The selection of appropriate cultureconditions, such as temperature and pH, are well within the skill of theordinarily skilled artisan.

As representative examples of appropriate hosts, include, but are notlimited to, bacterial cells, such as E. coli, Salmonella typhimurium,Streptomyces; fungal cells, such as yeast; insect cells, such asDrosophila S2 and Spodoptera Sf9; mammalian cells such as CHO, COS orBowes melanoma. The selection of an appropriate host is deemed to bewithin the scope of those skilled in the art from the teachings herein.

Introduction of the construct into the host cell can be effected, forexample, by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis et al., BASIC METHODS INMOLECULAR BIOLOGY, 1986). Constructs in host cells can be used in aconventional manner to produce the gene product encoded by therecombinant sequence.

Protein Expression

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described above and in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2d ed., (Cold Spring Harbor, N.Y., 1989).

Transcription of a DNA encoding polypeptides of the present invention byhigher eukaryotes can be increased by inserting an enhancer sequenceinto the expression vector. Enhancers are cis-acting elements of DNA,usually from about 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers. Generally, recombinant expression vectors willinclude origins of replication and selectable markers permittingtransformation of the host cell, e.g., the ampicillin resistance gene ofE. coli and S. cerevisiae TRP1 gene, and a promoter derived from ahighly expressed gene to direct transcription of a downstream structuralsequence. Such promoters can be derived from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), α factor, acidphosphatase, or heat shock proteins, among others. The heterologousstructural sequence is assembled in appropriate phase with translation,initiation and termination sequences, and preferably, a leader sequencecapable of directing secretion of translated protein into theperiplasmic space or extracellular medium. Optionally, the heterologoussequence can encode a fusion protein including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

After transformation of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction), and cells are cultured for an additional period. Cells aretypically harvested by centrifugation and disrupted by physical orchemical means. The resulting crude extract is retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

Various mammalian cell culture systems also can be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, C127, 3T3, CHO, HeLa and HBK cell lines.

Protein Purification

Polypeptides of the present invention may be recovered and purified fromrecombinant cell cultures by methods well known in the art, includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxyapatite chromatography, and lectin chromatography. Highperformance liquid chromatography (HPLC) can be employed as a finalpurification step.

Polypeptides of the invention can be conveniently isolated as describedin the specific examples below. A purified polypeptide is at least about70% pure, that is, the isolated polypeptide is substantially free ofcellular material and has less than about 30% (by dry weight) ofnon-polypeptide material. Preferably, the preparations are 85% through99% (i.e., 85, 87, 89, 91, 93, 95, 96, 97, 98, and 99%) pure. Purity ofthe preparations can be assessed by any means known in the art, such asSDS-polyacrylamide gel electrophoresis, mass spectroscopy and liquidchromatography.

Post-Translational Modification

Depending upon the host employed in a recombinant production procedure,polypeptides of the invention may be glycosylated with mammalian orother eukaryotic carbohydrates or may be non-glycosylated. Polypeptidesof the invention may also include an initial methionine amino acidresidue.

Chemical Synthesis

Alternatively, polypeptides of the invention can be produced usingchemical methods to synthesize its amino acid sequence, such as bydirect peptide synthesis using solid-phase techniques (see, for example,Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al,Science 269, 202-204, 1995). Polypeptide synthesis can be performedusing manual techniques or by automation. Automated synthesis can beachieved, for example, using Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer). Optionally, fragments of a polypeptide can be separatelysynthesized and combined using chemical methods to produce a full-lengthmolecule.

A newly synthesized polypeptide can be substantially purified bypreparative high performance liquid chromatography (see, for example,Creighton, Proteins: Structures And Molecular Principles, WH Freeman andCo., New York, N.Y., 1983). The composition of a synthetic polypeptideof the present invention can be confirmed by amino acid analysis orsequencing by, for example, the Edman degradation procedure (see,Creighton, supra). Additionally, any portion of the amino acid sequenceof the polypeptide can be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins toproduce a variant polypeptide or a fusion polypeptide.

Pharmaceutical Applications

Polypeptides of the present invention can be used to treat type 2diabetes (non-insulin dependent diabetes mellitus) and/or to preventsubjects with impaired glucose tolerance, impaired fasting glucose,hypoglycemia, and/or obesity from developing type 2 diabetes.

Pharmaceutical Compositions

Polypeptides of the present invention may be combined with a suitablepharmaceutical carrier to form a pharmaceutical composition forparenteral administration. Such compositions comprise a therapeuticallyeffective amount of the polypeptide and a pharmaceutically acceptablecarrier or excipient. Pharmaceutically acceptable carriers, include butare not limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The formulation should suit the modeof administration.

Pharmaceutical compositions may be administered in a convenient manner,e.g., by oral, topical, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, or intradermal routes. Pharmaceuticalcompositions are administered in an amount, that is effective fortreating and/or prophylaxis of the specific indication. Suitable dosesrange from at least about 3.5 ng of active polypeptide (i.e., notincluding the weight of a PEG or fatty acid moiety)/kg body weight toabout 100 μg/kg body weight per day. In most cases, the dosage is fromabout 0.1 μg/kg to about 35 μg/kg (i.e., 0.1, 1, 5, 10, 15, 20, 25, 30,and 35 μg/kg) body weight daily, taking into account the routes ofadministration, symptoms, etc. These numbers do not take into accountthe bioavailability of the peptide in vivo, in which case more or lessmay be used to attain the effective dose desired. Determination of adose is well within the skill of the ordinary artisan and requires onlyroutine screening.

Polypeptides of the present invention may also be employed incombination with other pharmaceutical agents useful in the treatment ofdiabetes, impaired fasting glucose, impaired glucose tolerance,hyperglycemia, and obesity. Suitable agents include insulinsecretagogues, insulin sensitizers, and metformin HCl.

Kits

The invention also provides pharmaceutical packs or kits comprising oneor more containers filled with one or more of the ingredients ofpharmaceutical compositions of the invention. A notice in a formprescribed by a governmental agency that regulates the manufacture, useor sale of pharmaceuticals or biological products and reflectingapproval by the agency can be associated with the pack or kit.

Gene Therapy

A polypeptide of the invention may also be expressed in vivo, which isoften referred to as “gene therapy.” Thus, for example, cells may beengineered with a polynucleotide (DNA or RNA) encoding for thepolypeptide ex vivo and the engineered cells then provided to a patientto be treated with the polypeptide. Such methods are well known in theart. For example, cells may be engineered by procedures known in the artby use of a retroviral particle containing RNA encoding for polypeptidesof the present invention.

In a preferred embodiment, the DNA encoding the polypeptides of theinvention is used in gene therapy for disorders such as diabetes.According to this embodiment, gene therapy with DNA encodingpolypeptides of the invention is provided to a patient in need thereof,concurrent with, or immediately after diagnosis.

Local delivery of polypeptides using gene therapy may provide thetherapeutic agent to the target area, i.e., the pancreas. For instance apancreas-specific promoter was used to create a beta-cell pancreatictumor mouse model (Hanahan, Nature 315(6015): 115-22, 1985).

Both in vitro and in vivo gene therapy methodologies are contemplated.Several methods for transferring potentially therapeutic genes todefined cell populations are known. See, e.g., Mulligan, Science 260:926-31, 1993. These methods include:

1) Direct gene transfer. See, e.g., Wolff et al, “Direct Gene transferInto Mouse Muscle In Vivo,” Science 247:1465-68, 1990;

2) Liposome-mediated DNA transfer. See, e.g., Caplen et al.,“Liposome-mediated CFTR Gene Transfer To The Nasal Epithelium OfPatients With Cystic Fibrosis,” Nature Med. 3: 39-46, 1995; Crystal,“The Gene As A Drug,” Nature Med. 1:15-17, 1995; Gao and Huang, “A NovelCationic Liposome Reagent For Efficient Transfection Of MammalianCells,” Biochem. Biophys. Res. Comm. 179:280-85, 1991;

3) Retrovirus-mediated DNA transfer. See, e.g., Kay et al., “In VivoGene Therapy Of Hemophilia B: Sustained Partial Correction In FactorIX-Deficient Dogs,” Science 262:117-19, 1993; Anderson, “Human GeneTherapy,” Science 256:808-13, 1992.

4) DNA Virus-mediated DNA transfer. Such DNA viruses includeadenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses(preferably herpes simplex virus based vectors), and parvoviruses(preferably “defective” or non-autonomous parvovirus based vectors, morepreferably adeno-associated virus based vectors, most preferably AAV-2based vectors). See, e.g., Ali et al., “The Use Of DNA Viruses AsVectors For Gene Therapy,” Gene Therapy 1:367-84,1994; U.S. Pat. No.4,797,368, and U.S. Pat. No. 5,139,941.

Gene Therapy Vector Systems

The choice of a particular vector system for transferring a gene ofinterest will depend on a variety of factors. One important factor isthe nature of the target cell population. Although retroviral vectorshave been extensively studied and used in a number of gene therapyapplications, these vectors are generally unsuited for infectingnon-dividing cells. In addition, retroviruses have the potential foroncogenicity. However, recent developments in the field of lentiviralvectors may circumvent some of these limitations. See Naldini et al.,Science 272:263-7, 1996.

The skilled artisan will appreciate that any suitable gene therapyvector encoding polypeptides of the invention can be used in accordancewith this embodiment. The techniques for constructing such vectors areknown. See, e.g., Anderson, Nature 392 25-30, 1998; Verma and Somia,Nature 389 239-242, 1998. Introduction of the vector to the target sitemay be accomplished using known techniques.

Suitable gene therapy vectors include one or more promoters. Suitablepromoters which may be used include, but are not limited to, viralpromoters (e.g., retroviral LTR, SV40 promoter, adenovirus major latepromoter, respiratory syncytial virus promoter, B19 parvovirus promoter,and human cytomegalovirus (CMV) promoter described in Miller et al.,Biotechniques 7(9): 980-990, 1989), cellular promoters (e.g., histone,pol III, and β-actin promoters), and inducible promoters (e.g., MMTpromoter, metallothionein promoter, and heat shock promoter). Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

Retroviruses

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus,and mammary tumor virus. In one embodiment, the retroviral plasmidvector is derived from Moloney Murine Leukemia Virus.

The retroviral plasmid vector is used to transduce packaging cell linesto form producer cell lines. Examples of packaging cells which maybetransfected include, but are not limited to, the PE501, PA317, Ψ-2,Ψ-AM, PA12, T19-14, VT-19-17-H2, ΨCRE, ΨCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, 1: 5-14,1990. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host. Theproducer cell line generates infectious retroviral vector particles thatinclude the nucleic acid sequence(s) encoding polypeptides of theinvention. Such retroviral vector particles then may be used, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encodingpolypeptides of the invention. Eukaryotic cells that can be transducedinclude, but are not limited to, embryonic stem cells, embryoniccarcinoma cells, as well as hematopoietic stem cells, hepatocytes,fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchialepithelial cells.

Adenoviruses and Adeno-Associated Viruses

Adenoviruses have the advantage that they have a broad host range, caninfect quiescent or terminally differentiated cells, such as neurons orhepatocytes, and appear essentially non-oncogenic. See, e.g., Ali etal., 1994, at page 367. Adenoviruses do not appear to integrate into thehost genome. Because they exist extrachromosomally, the risk ofinsertional mutagenesis is greatly reduced. Ali et al., 1994, at page373.

Adeno-associated viruses exhibit similar advantages as adenoviral-basedvectors. However, AAVs exhibit site-specific integration on humanchromosome 19 (Ali et al., 1994, at page 377).

Transkaryotic Therapy

A different approach to gene therapy is “transkaryotic therapy” whereinthe patient's cells are treated ex vivo to induce the dormantchromosomal genes to produce the protein of interest afterreintroduction to the patient. Transkaryotic therapy assumes theindividual has a normal complement of genes necessary for activation.Transkaryotic therapy involves introducing a promoter or other exogenousregulatory sequence capable of activating the nascent genes, into thechromosomal DNA of the patients' cells ex vivo, culturing and selectingfor active protein-producing cells, and then reintroducing the activatedcells into the patient with the intent that they then become fullyestablished. The “gene activated” cells then manufacture the protein ofinterest for some significant amount of time, perhaps for as long as thelife of the patient. U.S. Pat. Nos. 5,641,670 and 5,733,761 disclose indetail this concept.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purpose ofillustration only, and are not to be construed as limiting the scope ofthe invention in any manner. All patents, patent applications, andreferences cited in this disclosure are incorporated herein by referencein their entirety.

EXAMPLES Example 1

Peptide Synthesis Methodology

The following general procedure was followed to synthesize some ofpolypeptides of the invention. Peptide synthesis was carried out by theFMOC/t-Butyl strategy (Peptide Synthesis Protocols (1994), Volume 35 byMichael W. Pennington & Ben M. Dunn) under continuous flow conditionsusing Rapp-Polymere PEG-Polystyrene resins (Rapp-Polymere, Tubingen,Germany). At the completion of synthesis, peptides were cleaved from theresin and de-protected using TFA/DTT/H₂O/Triisopropyl silane (88/5/5/2).Peptides were precipitated from the cleavage cocktail using cold diethylether. The precipitate was washed three times with the cold ether andthen dissolved in 5% acetic acid prior to lyophilization. Peptideidentity was confirmed by reversed-phase chromatography on a YMC-PackODS-AQ column (YMC, Inc., Wilmington, N.C.) on a Waters ALLIANCE® system(Waters Corporation, Milford, Mass.) using water/acetonitrile with 3%TFA as a gradient from 0% to 100% acetonitrile, and by MALDI massspectrometry on a VOYAGER DE™ MALDI Mass Spectrometer, (model 5-2386-00,PerSeptive BioSystems, Framingham, Mass.). Matrix buffer (50/50dH₂O/acetonitrile with 3% TFA) peptide sample was added to Matrix buffer1/1. Those peptides not meeting the purity criteria of >95% werepurified by reversed-phase chromatography on a Waters Delta Prep 4000HPLC system (Waters Corporation, Milford, Mass.).

Example 2

Peptide Cloning

To establish a robust method for expressing polypeptides of theinvention and mutants thereof, nucleotide sequences encodingpolypeptides were cloned C-terminal to GST with a single Factor Xarecognition site separating the monomeric peptide and GST. The geneencoding the Factor Xa recognition site fused to the DNA sequence of thepeptide to be produced has been synthesized by hybridizing twooverlapping single-stranded DNA fragments (70-90mers) containing a BamHIor EcoRI restriction enzyme site immediately 5′ to the DNA sequence ofthe polynucleotide to be cloned, followed by DNA synthesis of theopposite strands via the large fragment of DNA polymerase I (LifeTechnologies, Inc., Gaithersburg, Md.).

The DNA sequence chosen for each polynucleotide was based on the reversetranslation of the designed amino acid sequence of each peptide. In somecases, the polynucleotide encoding the peptide is generated by PCRmutagenesis (Picard et al., Nucleic Acids Res 22: 2587-91, 1994;Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.,Cold Spring Harbor Laboratory Press, New York) of a polynucleotidealready made by the method described above. The double-stranded productis then digested by BamHI and EcoRI and ligated into pGEX-6P-1 (AmershamPharmacia Biotech) which has also been cleaved by BamHI and EcoRI.

For example, when the DNA sequence the polypeptide identified as SEQ IDNO:7 was cloned into pGEX-6P-1, the following polypeptide sequence wasexpressed as fusions with glutathione S-transferase (GST):IEGRHSQGTFTSDYAKYLDARRAKEFIAWLVKGRG (SEQ ID NO:33), where the first 4amino acids, IEGR, is the factor Xa recognition site and the remaining31 amino acids is the amino acid sequence identified as SEQ ID NO:7.

The first and last six nucleotides of the nucleic acid sequence encodingthe polypeptide identified as SEQ ID NO:7 represent the restrictionenzyme sites (BamHI and EcoRI) used for cloning into the pGEX6P-1plasmid (FIG. 1). The “TAATGA” sequence immediately preceding the EcoRIsite (“GAATTC”) encodes two stop codons. The rest of the DNA sequenceencodes the Factor Xa-polypeptide identified as SEQ ID NO:7 fusionsequence.

Example 3

Peptide Recombinant Expression and Purification

BL21 (DE3) cells (Stratagene) transformed with a GST-peptide fusioncontaining plasmids were grown at 37° C. until OD₆₀₀ reached 0.6 to 1.0and induced by 1 mM IPTG (Life Technologies) for 2 hours at 37° C. Twoliters of cells were spun at 7,700×g for 15 minutes, and stored at −20°C. The cell pellet was resuspended in 80 ml B-PER bacterial proteinextraction reagent (Cat No. 78248, Pierce) and 1× Complete ProteaseInhibitor (Roche) until the cell suspension was homogenous. Thehomogenous mixture was gently shaken at room temperature for 10 minutes.Lysosome and Dnase I were added to a final concentration of 200 μg/mland 10 μg/ml to further solubilize proteins and to reduce viscosity,respectively. The mixture was incubated for an additional 5 minutes.Cellular debris was spun down at 27,000 g for 20 minutes. Thesupernatant was mixed with 2 mL of pre-washed Glutathione Sepharose 4Bresin (Pharmacia) on a shaker overnight at 4° C. The resin was spun downat 1,500 g for 15 minutes, packed into empty Poly-Prep ChromatographyColumns (Bio-Rad), washed with 30 mL PBS followed by 10 mL of Factor Xabuffer (1 mM CaCl₂, 100 mM NaCl, and 50 mM Tris-HCl, pH 8.0). Thepeptides were cleaved off the column with 60 units of Factor Xa(Pharmacia) in 1 mL of Factor Xa buffer, overnight at 4° C. Theseeluants were then run on a C₁₈ HPLC (Beckman System Gold), using a 2 mLloop and flow rate of 1 mL/min with the following program: 5 minutes ofBuffer A (0.1% TFA/H₂0), 30 minutes of gradient to 100% Buffer B (0.1%TFA/ACN), 10 minutes of Buffer A, 10 minutes of gradient, and 10 minutesof Buffer A. Peak fractions (0.5 mL each) were collected and screened by10-20% Tricine-SDS gel electrophoresis. The identity of each peptide wasconfirmed by mass spectrometry using the mass predicted from the aminoacid sequence. Typical yields are approximately 50 μg free peptides perliter of E. coli culture. Recombinant peptides have been shown to havethe same activities as their synthetic versions.

The following table, Table 2, contains some selected polypeptides madeaccording to the peptide synthesis protocol discussed above (Example 1),or recombinantly as described above.

TABLE 2 Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 1) GLP-1(7–36) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (SEQ ID NO: 2) GLP-1 (7–37)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 3) G1   HSQGTFTSDYSKYLDSRRAQDFVQWLVKGR-NH2 (SEQ ID NO: 4) G5   HSQGTFTSDYSKYLEGQAAKEFIAWLVKGR-NH2 (SEQ ID NO: 5) G27  HSQGTFTSDYAKYLDARRAKEFIAWLVKGR-NH2 (SEQ ID NO: 6) G51  HSQGTFTSDYAKYLDARRAKEFIAWLVKGRG (SEQ ID NO: 7) G55  HSQGTFTSDYARYLDARRAKEFIAWLVKGR-NH2 (SEQ ID NO: 8) G56  HSQGTFTSDYAAYLDARRAKEFIAWLVKGR-NH2 (SEQ ID NO: 9) G57  HSQGTFTSDYAKYLDAARAKEFIAWLVKGR-NH2 (SEQ ID NO: 10) G58  HSQGTFTSDYAKYLDAKKAKEFIAWLVKGRG (SEQ ID NO: 11) G59  HSQGTFTSDYARYLDAKKAKEFIAWLVKGRG (SEQ ID NO: 12) G60  HSQGTFTSDYAKYLDAAKAKEFIAWLVKGRG (SEQ ID NO: 13) G61  HSQGTFTSDYARYLDAAKAKEFIAWLVKGRG (SEQ ID NO: 14) G71  HSQGTFTSDYAKYLDARRACEFIAWLVKGRG (SEQ ID NO: 15) G74  HSQGTFTSDYAKYLDARRAKEFIAWLVCGRG (SEQ ID NO: 16) G75  HSQGTFTSDYAKYLDARRAKEFIAWLVKCRG (SEQ ID NO: 17) G76  HSQGTFTSDYAKYLDARRAKEFIAWLVKGCG (SEQ ID NO: 18) G77  HSQGTFTSDYAKYLDARRAKEFIAWLVKGRC (SEQ ID NO: 19) G277 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRC (22 kD) (SEQ ID NO: 20) G82  HSQGTFTSDYARYLDARRAKEFIAWLVRGRG (SEQ ID NO: 21) G83  HSQGTFTSDYARYLDARRAREFIKWLVRGRG (SEQ ID NO: 22) G84  HSQGTFTSDYARYLDARRAREFIAWLVKGRG (SEQ ID NO: 23) G85  HSQGTFTSDYARYLDARRAREFIAWLVRGRGK (SEQ ID NO: 24) G185 HSQGTFTSDYARYLDARRAREFIKWLVRGRC (SEQ ID NO: 25) G2185HSQGTFTSDYARYLDARRAREFIKWLVRGRC (22 kD) (SEQ ID NO: 26) G4185HSQGTFTSDYARYLDARRAREFIKWLVRGRC (43 kD) (SEQ ID NO: 27) G87  HSQGTFTSDYAKYLDARRAK (FA) EFIAWLVKGR-NH2 (SEQ ID NO: 28) G88  HSQGTFTSDYAKYLDARRAKEFIAWLVKGRK (FA) (SEQ ID NO: 29) G90  HSQGTFTSDYARYLDARRAREFIK (FA) WLVRGRG (SEQ ID NO: 30) G182 HSQGTFTSDYARYLDARRAREFIKWLVRGRGK (FA) (SEQ ID NO: 31) G183 HSQGTFTSDYARYLDARRAK (FA) EFIKWLVRGRG (SEQ ID NO: 32) (FA): C₁₆palmitate linked to the lysine residue. (22 kD): 22 kD PEG linked to thecysteine residue. (43 kD): 43 kD PEG linked to the cysteine residue.

Example 4

Preparation of RINm5F Cell Membranes

Flasks of RINm5F cells were washed with PBS, scraped in 20 mM Hepes-1 mMEDTA-250 mM sucrose buffer containing protease inhibitors (HES) andhomogenized in a dounce homogenizer followed by repeated resuspensionthrough a 23 gauge needle. Unbroken cells and nuclei were removed bycentrifugation at 500×g for 5 minutes. The pellet was resuspended in HESbuffer using a 23 gauge needle and the centrifugation was repeated. Thesupernatants from the two spins were combined and centrifuged at40,000×g for 20 min. The resulting plasma membrane pellet wasresuspended in HES buffer using a 23 gauge needle followed by a 25 gaugeneedle. Membranes were stored at −80° C. until use.

Example 5

Preparation of Plasma Membranes from Rat Liver

Rats were sacrificed and livers removed into ice cold TES buffer (20 mMTris, pH 7.5, 1 mM EDTA, 255 mM sucrose containing protease inhibitors).The wet weight of the livers was determined and the tissue minced in 5volumes of ice cold TES buffer and a slurry prepared using a polytron.All buffers and spins were kept at 4° C. The slurry was furtherhomogenized using three strokes of a handheld dounce homogenizer. Thehomogenate was passed through several layers of cheesecloth and spun at25,000×g for 10 minutes. Pellets were homogenized in 22.1 ml TES bufferusing 10 strokes of a handheld dounce homogenizer. Twenty-seven pointnine milliliters of 2.4 M sucrose in TE was added and mixed well. Thehomogenate was distributed among several tubes, and each tube wasoverlaid with 7 ml of TES buffer. The tubes were spun at 120,000×g for60 minutes. The plasma membranes were removed from the interface thatformed during the spin, diluted in TES and concentrated using a 15minute spin at 200,000×g. The resulting pellet was again homogenized in9.8 ml TES buffer, combined with 10.3 ml 2.4M sucrose, overlaid with 7ml TES, and centrifuged at 180,000×g for 30 min. The plasma membraneswere removed from the interface, diluted with TES buffer, and collectedby centrifugation at 200,000×g for 15 min. The final pellet wasresuspended in TES. Plasma membranes were stored at −80° C. until readyfor use.

Example 6

Preparation of Rat Hepatocytes

Hepatocytes were isolated according to a modified procedure of Berry andFriend (J. Cell. Biol. 43:506, 1969). A fed male Wistar rat wasanesthetized with sodium pentobarbital (55 mg/kg, i.p.). The rat wasplaced on its back on the tray of the liver perfusion apparatus (37°C.), and its limbs were secured with laboratory tape. The ventricalsurface was cleaned with 70% alcohol, and the abdominal cavity wasopened with scissors to expose the liver, portal vein, hepatic arteryand the posterior vena cava. Ligatures were placed loosely around thethree vessels. A cannula was inserted into the portal vein and wassecured with the ligature. The hepatic artery and the posterior venacava below the liver were tied off with the ligatures. The perfusionpump was turned on and the descending aorta was immediately severed toallow the buffer to escape and to exsanguinate the rat. The chest cavitywas quickly opened to expose the heart. A cannula was inserted into thesuperior vena cava through the heart and was secured with a ligature tothe complete the circulation of buffer through the liver. The liver wasperfused with Krebs-Henseleit buffer containing 5-10% sheep RBC andunder constant 95% O₂/5% CO₂ flow in situ at a flow rate of 14ml/minute. It was perfused in a non-circulating system for 5 min andthen followed by a re-circulating system (with 30 mg collagenase) foranother 30 minutes. The liver was then removed, minced with scissors ina plastic beaker and filtered through a nylon sieve. The hepatocyteswere separated from debris by a series of buffer washings andcentrifugations (690 rpm, room temperature, 90 sec). An aliquot of cellswas diluted with buffer, stained with 0.4% Trypan Blue and counted. Therest of the cells were diluted with buffer to a density appropriate foreach assay.

Example 7

Protocol for Rat Islet Isolation

Sprague Dawley rats (275-320 g) were used as the source of donor islets.Briefly, the pancreas was filled with 10 ml of cold reconstitutedLiberase RI (Boehringer Manheim), harvested and incubated withadditional 5 ml enzyme solution in water bath for 30 minutes. Tissuesuspension was washed twice with cold 10% FBS/Hanks buffer (Gibco),resuspended in 8 ml 25% ficoll (Sigma) and then layered with 5 ml eachof 23%, 20% and 11% ficoll. The islets in the 20% layer aftercentrifugation were removed, washed twice with cold 10% FBS/Hank bufferand resuspended in 10% FBS/RPMI 1640 media (Sigma).

Example 8

Competitive Binding of Peptide to the GLP-1 Receptor in RINm5F CellPlasma Membranes

IC₅₀ values for the competitive binding of polypeptides and polypeptidefragments, variants, and analogs of the invention to the GLP-1 receptorin RINm5F membranes typically are at least about 0.01 nM up to about 20nM (i.e., 0.01, 0.1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nM). IC₅₀ values for polypeptide derivatives ofthe invention typically are at least 0.01 nM up to about 500 nM (i.e.,0.01, 0.1, 1, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500nM).

Competitive binding of some polypeptides of the invention to RINm5F cellplasma membranes was measured as follows. Ninety-six-well GF/Cfiltration plates (Millipore, Bedford, Mass.) were blocked with 0.3% PEIfor at least one hour and washed twice with binding buffer consisting of20 mM Tris, 2 mM EDTA, pH 7.5, 1 mg/ml BSA, and 1 mg/ml bacitracin. Fivemicrograms of RINm5F cell plasma membranes diluted in binding bufferwere applied to each well together with 0.05 μCi ¹²⁵I labeled GLP-1 andpeptide concentrations ranging from 1×10⁻¹² to 1×10⁻⁵ M. Following a 60minute incubation at room temperature, the plates were washed 3 timeswith ice-cold PBS containing 1 mg/ml BSA. The plates were dried,scintillant was added to each well, and cpm per well determined using aWallac Microbeta counter.

The number of ¹²⁵, counts bound to the membranes at each concentrationof peptide were plotted and analyzed by nonlinear regression using Prizmsoftware to determine the IC₅₀. The polypeptides disclosed in Table 2bound to the GLP-1 receptor present in the plasma membranes isolatedfrom RINm5F cells with IC₅₀ values of between 1.4 nM and 248 nM(determined from a minimum of three trials). IC₅₀ is the concentrationof a polypeptide at which maximal binding of labeled GLP-1 (7-36) (SEQID NO:2) is reduced by 50%.

Example 9

Competitive Binding of Peptide to the Glucagon Receptor in Rat LiverPlasma Membranes

IC₅₀ values for the competitive binding of polypeptides and polypeptidefragments, derivatives, variants, and analogs of the invention to theglucagon receptor in rat liver membranes typically are at least about0.1 nM up to about 1000 nM (i.e., 0.1 1, 10, 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or1000 nM).

Competitive binding of some polypeptides of the invention to rat liverplasma membranes was measured as follows. Ninety-six-well GF/Cfiltration plates (Millipore, Bedford, Mass.) were blocked with 0.1% PEIfor at least one hour and washed twice with binding buffer consisting of20 mM Tris, 2 mM EDTA, pH 7.5, 1 mg/ml BSA, and 1 mg/ml bacitracin.Three micrograms of rat liver plasma membranes diluted in binding bufferwere applied per well together with 0.05 μCi ¹²⁵I labeled glucagon andpeptide concentrations ranging from 1×10⁻¹² to 1×10⁻⁵ M. Following a 60minute incubation at room temperature, the plates were washed 3 timeswith ice-cold PBS containing 1 mg/ml BSA. The plates were dried,scintillant was added to each well, and cpm per well determined using aWallac Microbeta counter.

The number of ¹²⁵I counts bound to the membranes at each concentrationof peptide were plotted and analyzed by nonlinear regression using Prizmsoftware to determine the IC₅₀. The polypeptides disclosed in Table 2bound to the glucagon receptor present in the plasma membranes isolatedfrom rat liver with IC₅₀ values of between 11.7 nM and 726 nM(determined from a minimum of three trials). IC₅₀ is the concentrationof a polypeptide at which maximal binding of labeled glucagon is reducedby 50%.

Example 10

Measurement of Peptide Signaling Through GLP-1 Receptor Using Cyclic AMPScintillation Proximity Assay (SPA)

For polypeptides and polypeptide fragments, variants, and analogs of theinvention, “activation” of the GLP-1 receptor in a cAMP scintillationproximity assay is induction of a maximal activity that is at leastabout 80% up to about 200% (i.e., 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, or 200%) of the maximal activity induced by the native GLP-1 (7-36)(SEQ ID NO:2) with a relative potency of at least 4% up to about 1000%(i.e., 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, or 1000%). For polypeptide derivatives of theinvention, “activation” of the GLP-1 receptor in a cAMP scintillationproximity assay is induction of a maximal activity that is at leastabout 80% up to about 200% (i.e., 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, or 200%) of the maximal activity induced by the native GLP-1(7-36) (SEQ ID NO:2) with a relative potency of at least 0.5% up toabout 100.0% (i.e., 0.5, 1, 10, 50, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000%). “Relative potency” is the EC₅₀ of native GLP-1(7-36) (SEQ ID NO:2) divided by the EC₅₀ of a polypeptide of theinvention, multiplied by 100. “EC₅₀” is the concentration of apolypeptide at which 50% of the maximal activity is achieved.

Peptide signaling of GLP-1 receptor for some polypeptides of theinvention using cAMP scintillation proximity assay was measured asfollows. RINm5F cells were plated in 96-well plates (Costar) at1.5×10⁵cells/well and grown at 37° C. for 24 hours in RPMI 1640, 5% FBS,antibiotic/antimycotic (Gibco BRL). The media was removed and the cellswere washed twice with PBS. The cells were incubated with peptideconcentrations ranging from 1×10⁻¹² to 1×10⁻⁵ M in Hepes-PBS containing1% BSA and 100 μM IBMX for 15 min at 37° C. For assay of peptidesconjugated with fatty acid, the BSA was omitted from the incubationbuffer. The incubation buffer was removed, and the cells were lysed inthe lysis reagent provided with the cAMP Scintillation Proximity Assay(SPA) direct screening assay system (Amersham Pharmacia Biotech Inc,Piscataway, N.J.). The amount of cAMP (in pmol) present in the lysateswas determined following instructions provided with this kit.

The amount of cAMP (in pmol) produced at each concentration of peptidewas plotted and analyzed by nonlinear regression using Prizm software todetermine the EC₅₀ for each peptide. The average relative potency valuefor GLP-1 receptor activation for the polypeptides disclosed in Table 2was between 0.6% and 76.1%. The maximum activity induced ranged from 83%to 132% of the native peptide GLP-1 (7-36) (SEQ ID NO:2) (determinedfrom a minimum of three trials). See Table 4.

Example 11

Measurement of Peptide Signaling Through Glucagon Receptor Using CyclicAMP Scintillation Proximity Assay (SPA)

For polypeptides and polypeptide fragments, variants, and analogs of theinvention, “activation” of the glucagon receptor in a cAMP scintillationproximity assay is induction of a maximal activity that is at leastabout 0% up to about 75% (i.e., 0, 10 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, or 75%) of the maximal activity induced by the nativeglucagon (SEQ ID NO:1) with a relative potency from at least about 0.001to about 5% (i.e., 0.001, 0.01, 0.1, 1, 2, 3, 4, or 5%). For polypeptidederivatives of the invention, “activation” of the glucagon receptor in acAMP scintillation proximity assay is induction of a maximal activitythat is at least about 0% to about 40% (i.e., 0, 1, 10, 20, 30, or 40%)of the maximal activity induced by the native glucagon (SEQ ID NO:1)with a relative potency of at least about 0.001 to about 1% (i.e.,0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1%).“Relative potency” is the EC₅₀ of native glucagon (SEQ ID NO:1) dividedby the EC₅₀ of a polypeptide of the invention, multiplied by 100. “EC₅₀”is the concentration of a polypeptide at which 50% of the maximalactivity is achieved.

Peptide signaling of glucagon receptor for some polypeptides of theinvention using cAMP scintillation proximity assay was measured asfollows. Freshly isolated rat hepatocytes were plated in 96 well platesat 7.5×10⁴ or 2×10⁵ cells/well in Hepes-bicarbonate-PBS containing 1%BSA and 100 μM IBMX. Following equilibration at 37° C. in a 5% CO₂/95%O₂ environment for 10 min, peptide at concentrations ranging from1×10⁻¹² to 1×10⁻⁵ M was added for an additional 15 min. The plates werecentrifuged briefly, the incubation buffer was removed, and the cellswere lysed in lysis reagent provided with the cAMP ScintillationProximity Assay (SPA) direct screening assay system (Amersham PharmaciaBiotech Inc, Piscataway, N.J.).

The amount of cAMP (in pmol) present in the lysate was determinedfollowing instructions provided with this kit. The amount of cAMP (inpmol) produced at each concentration of peptide was plotted and analyzedby nonlinear regression using Prizm software to determine the EC₅₀ foreach peptide. The average relative potency value (ratio of the glucagonconcentration to polypeptide concentration at 50% response (EC₅₀)×100,determined from a minimum of three trials) for glucagon receptoractivation for polypeptides of the invention was <5%. The maximumactivity induced ranged from 28.3% to 71.3% of the native peptideglucagon (determined from a minimum of three trials).

The ability of the hybrid peptide to inhibit glucagon activity wasmeasured as follows: Following equilibration at 37° C. in a 5% CO₂/95%O₂ environment for 10 min, 10 μM peptide was added to the cells followedimmediately by a submaximal concentration of glucagon for 15 min. Thecells were lysed and cAMP determined as described above.

After subtracting the amount of cAMP produced in the unstimulatedhepatocytes from each data point, the percent inhibition was calculatedas follows: Percent inhibition is the amount of cAMP produced in thepresence of submaximal glucagon alone less the amount of cAMP producedin the presence of submaximal glucagon and 10 μM peptide, divided by theamount of cAMP produced in the presence of submaximal glucagon alone andmultiplied by 100. The average percent inhibition of glucagon activityby the polypeptides disclosed in Table 2 was between 11.3% and 59%(determined from a minimum of three trials). See Table 4.

Example 12

Measurement of Glucose Release from Rat Hepatocytes

Inhibition of glucagon-mediated glucose production as measured byglucose release from rat hepatocytes is typically at least about 20%inhibition to about 100% inhibition (i.e., 20, 30, 40, 50, 60, 70, 80,90, or 100%).

Inhibition of glucagon mediated glucose production by some polypeptidesof the invention was measured as follows. Rat hepatocytes were addedinto a flat bottom 96 well plate (2×10⁵/100 μl/well) and pre-incubatedin a 37° C. incubator with constant shaking and under 95% O₂/5% CO₂ flowfor 10 minutes. Hepatocytes were incubated for another 30 minutes afteraddition of glucagon with or without peptide. Cells were then lysed with15% perchloric acid and plates were spun at 2600 rpm, 4° C. for 15 min.The supernatant was neutralized with 1M Tris-HCl (pH 8.0): 2.5 N KOH(45:55) and spun again. The resulting supernatant was analyzed forglucose with hexokinase and glucose-6-phosphate dehydrogenase (Methodsof Enzymatic Analysis, H. U. Bermeyer, Ed., Academic Press) and the A₃₄₀read on a fMAX plate reader (Molecular Devices, Sunnyvale, Calif.).

Glucose output was calculated after subtracting the amount of glucoseproduced in the unstimulated hepatocytes from each data point and thepercent inhibition was calculated. Percent inhibition is the amount ofglucose produced in the presence of 1 nM glucagon alone less the amountof glucose produced in the presence of 1 nM glucagon and 100 nM peptide,divided by the amount of glucose produced in the presence of 1 nMglucagon alone and multiplied by 100. The polypeptide having the aminoacid sequence shown in SEQ ID NO:6 inhibited glucagon mediated glucoseproduction by 47% as determined from 3 trials. See Table 2.

Example 13

Measurement of Insulin Secretion by Perfused Rat Islets

Increase of insulin secretion by perfused rat islets in this assay is anincrease of at least 1.5-fold. The GLP-1 receptor agonist component ofpolypeptides of the invention increases insulin secretion from perfusedislets by at least 1.5-fold to about 10-fold (i.e., 1.5, 2.0, 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold).

Insulin secretion of perfused rat islets mediated by some peptides ofthe invention was measured as follows. The bi-phasic responses ofinsulin release stimulated by these polypeptides were tested by isletperfusion. Fifty islets were loaded in the perifusion chamber andperifused with HEPES-KRB containing 3 mM glucose at 37° C. After 60 min,islets were exposed to buffer containing 8 mM glucose with or withoutpeptide (50 nM) and perifused for another 30 min. Fractions ofperifusate were collected at 1 or 5 minute intervals for insulindetermination. Insulin was measured using an ELISA kit (AlpcoDiagnostics, Windham, N.H.). At a concentration of 50 nM, thepolypeptide having the amino acid sequence shown in SEQ ID NO:6increased insulin secretion from perfused islets approximately 2-fold,which is equivalent to that achieved by 50 nM GLP-1.

Example 14

Insulin Secretion from Dispersed Rat Islet Cells

Increase of insulin secretion from dispersed rat islet cells, in thisassay, is an increase of at least 1.5-fold. The GLP-1 receptor agonistcomponent of such polypeptides of the invention increases insulinsecretion from dispersed islet cells by at least 1.5-fold to about10-fold (i.e., 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold).

Insulin secretion of dispersed rat islets mediated by a number ofpeptides of the invention was measured as follows. Islets of Langerhanswere isolated from SD rats (200-250 g) through a digestion procedureusing collagenase. Dispersed islet cells were prepared through treatmentwith trypsin, seeded into 96 V-bottom plates and pelleted. Cells werecultured overnight in media. Media was aspirated and the cells werepre-incubated with Krebs-Ringer-HEPES buffer containing 3 mM glucose for30 minutes at 37° C. Pre-incubation buffer was removed and cells werestimulated with Krebs-Ringer-HEPES buffer containing the appropriateglucose concentration (e.g., 8 mM), with and without peptides for anappropriate time at 37° C. In some studies, an appropriate concentrationof GLP-1 also is included. A portion of supernatant was removed and itsinsulin content was measured by SPA. The results are expressed as foldover control (FOC). At a concentration of 50 nM, the polypeptide havingan amino acid sequence shown in SEQ ID NO:27 increased insulin secretionfrom dispersed islet cells approximately 3-fold. See Table 2.

Example 15

Measuring Glucagon-Stimulated Glucose Production in Fed Wistar Rats

Inhibition of glucagon-stimulated glucose production in this assay is aninhibition of at least 20%. The glucagon receptor antagonist componentof polypeptides of the invention inhibits glucagon-mediated elevation inblood glucose as measured by glucagon stimulated glucose production infed Wistar rats is at least 20% inhibition to about 100% inhibition(i.e., 20, 30, 40, 50, 60, 70, 80, 90, or 100%).

Glucagon stimulated glucose production in fed Wistar rats by somepolypeptides of the invention was measured as follows. Male Wistar ratswere briefly anesthetized with isoflurane gas, tail bled for bloodglucose using a Glucometer, and then given an injection into the tailvein of either vehicle (0.9% saline+1% human albumin), 0.29 nmol/kgglucagon, 1 nmol/kg polypeptides of the invention orglucagon+polypeptides of the invention. The anesthesia was withdrawn andthe conscious rats were tail-bled again after 10, 20 and 30 min.

Glucose inhibition was determined by analyzing the area under theglucose curve after subtracting the basal glucose level (dAUC). Percentinhibition is defined as the amount of glucose produced by 0.3 nmol/kgglucagon alone less the amount of glucose produced by 0.3 nmol/kgglucagon in the presence of 1 nmol/kg hybrid peptide, divided by theamount of glucose produced by 0.3 nmol/kg glucagon alone and multipliedby 100.

Glucagon alone elevated blood glucose levels whereas polypeptides of theinvention alone had no effect on blood glucose levels. The polypeptidehaving the amino acid sequence shown in SEQ ID NO:6 inhibitedglucagon-mediated elevation in blood glucose by 63%. See Table 2.

Example 16

Measuring Glucagon-Stimulated Glucose Production in Fed Balb/C Mice

Inhibition of glucose production in this assay is an inhibition of atleast about 20%. Preferably, the glucagon receptor antagonist componentof polypeptides of the invention inhibits glucagon-mediated elevation inblood glucose in mice as measured by glucagon-stimulated glucoseproduction in fed Balb/C mice by about 20% to about 100%, (i.e., 20, 30,40, 50, 60, 70, 80, 90, or 100%).

Glucagon stimulated glucose production in fed Balb/C mice by somepolypeptides of the invention was measured as follows. Fed male Balb/Cmice were given vehicle (0.9% saline+1% human albumin) or 100 μg/kgderivatized polypeptide by subcutaneous injection 17 hours prior to theglucagon challenge. The next day the mice were fasted for 2 hours beforereceiving an intravenous injection of 10 μg/kg glucagon or vehicle inthe tail vein. The mice were tail bled for glucose using a Glucometerjust prior to the glucagon injection and again 15 minutes afterward.

The change in glucose over the 15 minutes was calculated for each mouse.Then the average change in glucose in the vehicle-treated group wassubtracted from the change in glucose for each mouse in the treatedgroups to obtain the change in glucose due to glucagon. Percentinhibition is defined as the amount of glucose produced by 10 μg/kgglucagon alone less the amount of glucose produced by 10 μg/kg glucagonin the presence of 100 μg/kg hybrid peptide, divided by the amount ofglucose produced by 100 μgl/kg glucagon alone and multiplied by 100. SeeTable 3

TABLE 3 Glucose (mg/dl) 15 Delta −Vehicle % of 10 μg/kg % Group 0 minmin (15 − 0) delta Glucagon Inhibition Vehicle 92 108 16  10 μg/kgglucagon 95 195 100 84 100  10 μg/kg glucagon + 75 150 75 59 69 31 100μg polypeptide (SEQ ID NO: 27)

Example 17

Measuring Increases in Plasma Insulin Levels During In Vivo GlucoseTolerance Testing (IVGTT) in Fasted Wistar Rats

An increase in plasma insulin levels in this assay is an increase of atleast about 2-fold. Preferably, the GLP-1 receptor agonist component ofpolypeptides of the invention increases insulin secretion in rats asmeasured by an increase in plasma insulin levels during in vivo glucosetolerance testing in fasted Wistar rats by about 2-fold to about 5-fold,more preferably by about 2-fold to about 10-fold, and still morepreferably by about 2-fold to about 20-fold (i.e., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-fold).

Plasma insulin levels during in vivo glucose tolerance testing in fastedWistar rats by some polypeptides of the invention were measured asfollows. Male Wistar rats were fasted overnight and then anesthetizedwith isoflurane gas. The rats were given a tail vein injection of 0.4g/kg of glucose plus either vehicle (0.9% saline+1% albumin) or 1nmol/kg GLP-1 (positive control) or 1 nmol/kg of polypeptides of theinvention. The rats were eye-bled one minute later and the plasmaassayed for insulin using an ELISA Kit (Alpco Diagnostics (Windham,N.H.)). At a concentration of 1 nmol/kg, the polypeptide having theamino acid sequence shown in SEQ ID NO:6 promoted insulin secretion 3-4fold, which is equivalent to that achieved by 1 nmol/kg GLP-1. See Table2.

Example 18

Effect of Peptides of the Invention on Intraperitoneal Glucose ToleranceTesting (IPGTT) in Rats or Mice

A decrease in blood glucose levels as measured by this assay is adecrease of at least about 10%. Preferably, the GLP-1 receptor agonistcomponent of polypeptides of the invention decreases blood glucoselevels in rats or mice as measured by intraperitoneal glucose tolerancetesting in rats or mice by about 10% to about 60%, more preferably byabout 10% to about 80%, still more preferably by about 10% to about 100%(i.e., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%).

Blood glucose levels during intraperitoneal glucose tolerance testing inrats or mice by some polypeptides of the invention were measured asfollows. The in vivo activity of the polypeptides of the invention whenadministered subcutaneously was examined in rats or mice. Rats or micefasted overnight were given a subcutaneous injection of control orpeptide (100 μg/kg). Basal blood glucose was measured prior toadministration of peptides or three or 17 hours after administration ofderivatized peptides, and the rats or mice were given 2 g/kg of glucoseintraperitoneally. Blood glucose was measured again after 15, 30 and 60minutes in rats or 30 and 60 minutes in mice.

Peptides of the invention significantly reduced blood glucose levelsrelative to the vehicle following the IPGTT, with 13%-54% reduction inthe glucose AUC. This demonstrates that peptides have prolonged glucoselowering activity and a prolonged half-life in vivo. GLP-1 has a veryshort half-life in vivo (<10 min.). The ability of the peptides of theinvention to lower blood glucose 3 hours following peptideadministration is a clear indication that the peptide is present in thecirculation at this time point and hence has prolonged half-liferelative to GLP-1.

Example 19

Peptide PEGylation

PEGylation can be performed by any method known to those skilled in theart. However, in this instance, PEGylation was performed by introducinga unique cysteine mutation into the peptide followed by PEGylating thecysteine via a stable thioether linkage between the sulfhydryl of thepeptide and maleimide group of the methoxy-PEG-maleimide reagent(Inhale/Shearwater). It is preferable to introduce the unique cysteineat the C-terminus of the peptide to minimize potential reduction ofactivity by PEGylation.

Specifically, a 2-fold molar excess of mPEG-mal (MW 22 kD and 43 kD)reagent was added to 1 mg of peptide (e.g., SEQ ID NO:25 having acysteine mutation at the C-terminus of the peptide) and dissolved inreaction buffer at pH 6 (0.1M Na phosphate/0.1M NaCl/0.1M EDTA). After0.5 hour at room temperature, the reaction was stopped with 2-fold molarexcess of DTT or free cysteine to mPEG-mal. The peptide-PEG-mal reactionmixture was applied to a cation exchange column to remove residual PEGreagents followed by gel filtration column to remove residual freepeptide. The purity, mass, and number of PEGylated sites were determinedby SDS-PAGE and MALDI-TOF mass spectrometry. PEGylation with a smallerPEG (e.g., a linear 22 kD PEG) will less likely reduce activity of thepeptide, whereas a larger PEG (e.g., a branched 43 kD PEG) will morelikely reduce activity. However, the larger PEG will increase plasmahalf life further so that once a week injection may be possible (Harris,et al., PEGylation: A Novel Process for Modifying Pharmacokinetics,Clin. Pharmacokinet 40:539-551, 2001).

Example 20

Relative Potency of GLP-1 Receptor Agonist and Glucagon ReceptorAntagonist Polypeptides

GLP-1 binding (IC₅₀ values) for polypeptides listed in Table 4 wasdetermined as described in Example 8. Relative potency values andpercent GLP-1 activity were calculated as described in Example 10. Ratsor mice were dosed with 100 μg/kg peptide and 3 hours post-dosing thedecrease in glucose levels were determined by intraperitoneal glucosetolerance testing (IPGTT) as described in Example 18. See Table 4.

Liver membrane binding (IC₅₀ values) for polypeptides listed in Table 4were determined as described in Example 9. Percent inhibition ofglucagon stimulated cAMP increase was determined as described in Example11. Rats or mice were dosed with 100 μg/kg peptide and 17 hourspost-dosing glucose levels were determined by the methods of Examples 15and 16 and reported in Table 4. Polypeptides of the invention functionas GLP-1 receptor agonists and glucagon receptor antagonists in vivo.

TABLE 4 Glucagon GLP1 In vivo In vivo % Inhib % Decrease Decreaseglucagon glucagon RIN Relative IpGTT Liver stimulated stimulated IC50Potency AUC IC50 cAMP blood peptide (nM) (%) % GLP1 max (%)^(a) (nM)increase glucose^(b) Glucagon 1.8 GLP-1 0.4 100 100 G1 6 6.9 112.1 6.6G5 0.5 69.9 109.4 243.1 G27 2.2 13.4 98.1 69.3 31 G51 8.2 58.1 91.2154.1 35.2 G55 9.8 18.6 115.9 139.2 32.1 G56 6.6 37.2 114.8 125.3 28.6G57 14.7 18.3 132.4 177.5 18.4 G58 6.9 76.1 99.0 163.5 32.6 G59 2.1 22.7101.7 97.5 41.1 G60 13.8 22.3 95.7 682 21.8 G61 10.6 12.1 96.0 232 45.2G71 5.8 4.3 93.3 25.6 11.3 G74 4.4 8.4 88.7 21.7 23.7 G75 7.7 6.6 86.154.0 17.1 G76 10.2 7.7 85 59.1 23.0 G77 8.9 9.5 92.1 61.1 18.6 G277 ND1.8 93.7 46 65.5 G82 8.1 29.9 97.1 33.6 30.2 G83 3.2 29.9 89.4 28.6 24.7G84 1.4 61.8 113.9 11.7 25.2 G85 1.5 64.5 95.8 16.1 27.9 G185 4.5 13.0103.9 48 32 G2185 56.1 6.2 112.7 44 347 G4185 248 0.7 98.3 36 547 31 G8798.6 6.1 100.4 13 620 59 G88 109.0 2.3 118.3 16 350 G90 25.4 0.8 83.5 13726 G182 12.6 1.4 104.4 14 220 G183 158.7 0.6 90.5 27 663 ^(a)= ipGTT inmice 3 hours post dosing with 100 μg/kg peptide ^(b)= glucagon challengein mice 17 hours post dosing with 100 μg/kg peptide ND = not determined

1. A method of treating a metabolic disorder associated with type 2diabetes in a mammal, comprising administering to the mammal atherapeutically effective amount of the polypeptide of SEQ ID NO:25,wherein said metabolic disorder is selected from the group consisting ofimpaired glucose tolerance, impaired fasting glucose, and obesity. 2.The method of claim 1, wherein said therapeutically effective amountranges from about 0.1 μg/kg to about 1 mg/kg.
 3. The method of claim 1,wherein said peptide is PEGylated.
 4. The method of claim 2, whereinsaid peptide is PEGylated.